Recombinant Mouse Mast Cell Protease-11/Prss34 Protein, CF

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Product Details

Summary
Reactivity MuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

Order Details

Recombinant Mouse Mast Cell Protease-11/Prss34 Protein, CF Summary

Details of Functionality
Measured by its ability to cleave a colorimetric peptide substrate, N-carbobenzyloxy-Arg-ThioBenzyl ester (Z-R-SBzl), in the presence of 5,5’Dithio-bis (2-nitrobenzoic acid) (DTNB). Edwards, K.M. et al. (1999) J. Biol. Chem. 274:30468. The specific activity is >20,000 pmol/min/µg, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived mouse Mast Cell Protease-11/Prss34 protein
Met20-Ser318, with a C-terminal 10-His tag
Accession # Q80UR4
Accession #
N-terminal Sequence
Met20
Structure / Form
Pro form
Protein/Peptide Type
Recombinant Enzymes
Gene
Prss34
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Theoretical MW
34 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
48 kDa doublet, reducing conditions

Packaging, Storage & Formulations

Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
Buffer
Supplied as a 0.2 μm filtered solution in MES and NaCl.
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Assay Procedure
  • Activation Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl2, pH 7.5 (TCN)
  • Assay Buffer: 50 mM Tris, pH 8.0
  • Recombinant Mouse Mast Cell Protease‑11/Prss34 (rmMCP-11) (Catalog # 2857-SE)
  • Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
  • 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
  • Substrate: Z-Arg-SBzl (MP Bio, Catalog # SB001), 10 mM in DMSO
  • 5,5’Dithio-bis(2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D-8130)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rmMCP-11 to 200 µg/mL with Activation Buffer.
  2. Dilute Thermolysin to 0.4 µg/mL with Activation Buffer.
  3. Mix equal volumes of 200 µg/mL rmMCP-11 and 0.4 µg/mL Thermolysin for final concentrations of 50 µg/mL and 0.2 µg/mL, respectively.
  4. Incubate at 37 °C for 30 minutes.
  5. Stop the reaction with 10 mM 1,10 Phenanthroline.
  6. Dilute activated rmMCP-11 to 0.1 ng/µL in Assay Buffer.
  7. Dilute substrate to 200 µM in Assay Buffer with 200 µM of DTNB.
  8. Load 50 µL of the 0.1 ng/µL rmMCP-11 into plate, and start the reaction by adding 50 µL of the substrate/DTNB mixture to wells. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL substrate mix without any rmMCP-11.
  9. Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
  10. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol
ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)

     *Adjusted for Substrate Blank
      **Using the extinction coefficient 13260 M-1cm-1
      ***Using the path correction 0.32 cm
     Note: the output of many spectrophotometers is in mOD Per Well:
  • rmMCP-11: 0.005 µg
  • DTNB: 100 µM
  • Substrate: 100 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse Mast Cell Protease-11/Prss34 Protein, CF

  • Mast Cell Protease11
  • Mast Cell Protease-11
  • MCP-11
  • MCPT11
  • Prss34

Background

Mast Cell Protease-11 (MCP-11) is encoded by Prss34, one of 13 genes on mouse chromosome 17A3.3 that correspond to functional trypsin-like serine proteases (1). The deduced amino acid sequence of mouse MCP-11 consists of 318 residues with a signal peptide (residues 1 to 19), a pro region (residue 20 to 34), and a catalytic domain (35 to 318). The mRNA is preferentially expressed in spleen and bone marrow. The mouse MCP-11 (residues 20 to 318) was expressed in the NS0 cells with a foreign signal peptide. After being treated with thermolysin, the purified enzyme is active against a peptide substrate described in the Activity Assay Protocol. Apparently, the human gene corresponding to Prss34 encodes a protein that is not enzymatically active due to a mutation that leads to a premature translation termination codon.

  1. Wong, G.W. et al. (2004) J. Biol. Chem. 279:2438.

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Bioinformatics

Gene Symbol Prss34
Uniprot